Method for making a steroid water soluble

ABSTRACT

A complex of branched beta cyclodextrin and steroid is formed by mixing a steroid and a branched beta cyclodextrin together in water for a period of about 4 to 24 hours under ambient conditions. The solubility of the steroid is increased about 95 times by employing the complex.

This is a continuation of application Ser. No. 920,010 filed Jul. 27,1992, now U.S. Pat. No. 5,229,370 which in turn is a continuation ofapplication Ser. No. 711,108 filed Jun. 18, 1991, now abandoned, whichin turn was a continuation of application Ser. No. 232,440 filed Aug.15, 1988, now abandoned.

This invention relates to a complex of branched beta cyclodextrin andsteroid and the use of such a complex to make the steroid water soluble.

Starch occurs naturally in a variety of plants such as corn, potato,sorghum and rice and is extracted from portions of the plant by amilling operation which separates the starch from the plant. Physicallythe starch is in a granular form which typically comprises both amyloseand amylopectin. Amylose is a straight chained polymer of anhydroglucoseunits bonded together by alpha 1,4 bonds while amylopectin is a polymercomposed of a straight chain of alpha 1,4 anhydroglucose onto which sidechains of alpha 1,4 anhydroglucose polymers are bonded. In amylopectin,the bond between the straight chain and the side chain is an alpha 1,6bond. The amount of amylose and amylopectin in a starch granule dependson the source of the starch. For example, high amylose corn starchcontains about a 50:50 ratio while waxy corn starch contains about a99:1 ratio of amylopectin to amylose.

Cyclodextrins, also called Schardingers dextrins, cycloamyloses,cyclomaltoses and cycloglucans, are polymers of anhydroglucose polymers,bonded together by alpha 1,4 bonds to form a ringed compound. A sixmembered ring is called alpha cyclodextrin, seven, beta cyclodextrin andeight, gamma cyclodextrin. These six, seven and eight membered rings arealso referred to as cyclomaltohexaose, cyclomaltoheptaose andcyclomaltooctaose, respectively.

Branched cyclodextrins were described as early as 1965 by French and hisco-workers, see French et al., "Archives of Biochem. and Biophys.",Volume III, 1965, pages 153-150, but had been studied very little untilrecently. Branched beta cyclodextrins, as their name implies, have oneor more anhydroglucose units bonded onto the ring structure such that abranch extends out from the ring structure. The branch is bonded to thering by an alpha 1,6 bond where the branched beta cyclodextrin isobtained from an enzymatic process.

Steroids are typically used in aqueous media and ointments aspharmaceutical preparations for treatment of the skin and eyes. Becausesteroids are highly water insoluble, any aqueous medium containingsteroids must be vigorously shaken before application. Steroids areclassified as lipids because they are highly water insoluble and aresoluble in organic solvents.

In order to increase the water solubility of the steroid, it has beensuggested in U.S. Pat. No. 4,383,992 issued May 17, 1983 that a complexof beta cyclodextrin and steroid be formed. It is alleged that such acomplex is water soluble. Typically, a complex of a steroid andunsubstituted beta cyclodextrin contains less than about 1/2% solublesteroid at ambient conditions.

It has also been suggested that in order to increase the watersolubility of a steroid a complex of alpha or gamma cyclodextrin andsteroid be formed. Under ambient conditions, the alphacyclodextrin-steroid solution typically contains less than about 1%soluble steroid and the gamma cyclodextrin-steroid solution alsotypically contains less than about 1% soluble steroid.

It has further been suggested that the beta cyclodextrin be chemicallymodified by reacting the beta cyclodextrin with methyl groups to formdimethyl beta cyclodextrin and then form a complex with a steroid. Sucha chemically modified starch is not ideal for pharmaceutical use.

It has now been discovered that branched beta cyclodextrin will form acomplex with a steroid and that such a complex is highly water soluble.In fact, it has been found that such a complex may have a solubility upto about 2800 times greater than that of the steroid itself and about100 times greater than that of the complex of beta cyclodextrin andsteroid.

These results are surprising because there had been speculation that thebranch of anhydroglucose might interfere with or in some way react withthe steroid itself and thus inhibit the complexation phenomenon betweenthe steroid and the cavity of branched beta cyclodextrin. This isespecially true for branched beta cyclodextrin where the branch lengthwas greater than one anhydroglucose unit. Additionally, it is surprisingthat the complex of branched beta cyclodextrin and steroid has asolubility comparable to a complex of dimethyl-beta cyclodextrin andsteroid. This is surprising because of the difference in chemicalstructure between the branched beta cyclodextrin and dimethyl-betacyclodextrin.

Further, it is surprising that a complex forms between branched betacyclodextrin and a steroid because of the hydrophilic nature of thebranch itself and the hydrophobic nature of steroids.

Further, it is surprising that crystals of branched beta cyclodextrinand steroid were formed where the branch is one anhydroglucose unitbecause branched beta cyclodextrin having a branch of one anhydroglucoseunit does not form a crystalline structure alone.

Branched beta cyclodextrins are conventionally prepared by treatingstarch slurry high in amylopectin with an enzyme, cyclodextringlycosyltransferase (CGT), at the appropriate pH, temperature and timefor the selected CGT. The starch used for this enzymatic preparation ofbranched beta cyclodextrin may be from any selected plant variety;however, it is preferable that the starch be high in amylopectincontent. The enzyme CGT is obtained from microorganisms such as Bacillusmacerans, B. megaterium, B. circulans, B. stearothermophilus andBacillus sp. (alkalophilic) as well as others. Conventionally, thestarch is slurried in aqueous solution at a concentration up to about35% by weight solids. The slurry is then subjected to gelatinization andliquefaction by enzyme or acid to about 1 to about 5 DE. The preferredenzyme for liquefaction is bacterial alpha amylase. After deactivatingthe liquefying enzyme, the selected CGT is added to the gelatinized andliquefied slurry and the pH, temperature and time of the treatment areadjusted depending on the selected enzyme. Generally, the pH is betweenabout 4.5 to about 8.5, the temperature ranges between ambient to about75° C. and the length of the reaction runs for about ten hours to sevendays. The amount of branched beta cyclodextrin will vary depending onthe treatment conditions and the starch selected.

In order to produce predominantly beta cyclodextrin and branched betacyclodextrin, the reaction between CGT and the gelatinized and liquefiedstarch slurry is conducted under a solvent such as toluene or p-xylene.Such solvents substantially facilitate the isolation of branched betacyclodextrin.

Normally, when starch is treated with CGT, branched beta cyclodextrins,beta cyclodextrins and other dextrins are formed. In order to obtainbranched beta cyclodextrin, the branched beta cyclodextrin must beseparated from the beta cyclodextrin and the other dextrins.

The purification and separation of branched beta cyclodextrin can besuccessfully accomplished by employing the method taught in U.S. Pat.No. 4,840,679 dated Jun. 20, 1989, which is incorporated by referenceherein. As taught by that reference, branched beta cyclodextrin isseparated from beta cyclodextrin and other dextrins by forming a firstprecipitate and a first liquor from an aqueous first solution containingbranched beta cyclodextrin by the addition of a beta cyclodextrincomplexant to the aqueous first solution; recovering the firstprecipitate; forming a second aqueous solution with the firstprecipitate; forming a second precipitate and a second liquor from saidsecond solution by the addition of a beta cyclodextrin complexant tosaid second solution; recovering the second liquor; and, finally,recovering branched beta cyclodextrin from said second liquor. Theaqueous starting solution is an aqueous solution containing branched andnon-branched beta cyclodextrins such that about 1% or more of the totalsolids content of the solution is branched beta cyclodextrin and suchthat the non-branched beta cyclodextrin is in an amount in excess of thebranched beta cyclodextrin. Invariably, some beta cyclodextrin will bepresent with the branched beta cyclodextrin.

Branched beta cyclodextrins are characterized more specifically by thelength of or the number of glucose units in the branch. For example, abranched beta cyclodextrin which has a single glucose unit attached tothe ringed structure is called 6-alpha-D-glucosylcyclomaltoheptaose orG1cG7, a branched beta cyclodextrin having two glucose units on thebranch is referred to as 6-alpha-D-maltosylcyclomaltoheptaose or G2cG7.A branched beta cyclodextrin with three glucose units on the branch isreferred to conveniently as G3cG7, four glucose units as G4cG7, and soon.

The branched beta cyclodextrins may also be formed by a pyrolysismethod, preferably as taught in the U.S. Pat. No. 5,007,967 issued Apr.16, 1991 Such branched beta cyclodextrins have branches ofanhydroglucose units which are bonded to the ring structure of the betacyclodextrin by bonds other than a 1,6 bond. Branched beta cyclodextrincan also be formed by enzymatic techniques where anhydroglucose unitsare added onto the cyclodextrin ring, see for example U.S. Pat. No.4,668,626 issued May 26, 1987. The term "branched beta cyclodextrin" asused in the specification and claims means branched beta cyclodextrinobtained from enzyme or pyrolysis.

Preferably, the branch length of the branched beta cyclodextrins used inthe present invention is between about 1 to about 7 glucose units, i.e.G1cG7 to G7cG7. Good results have been obtained with G1c7 to G3cG7.

It is thought that a complex forms between the steroid and the branchedbeta cyclodextrin where the steroid is a guest molecule inside thecavity of the branched beta cyclodextrin, which is acting as a hostmolecule. It is thought that not all of the steroid can physically fitinside the cavity of the branched beta cyclodextrin but that a portionof the polycyclic ring structure fits therein. Upon complexing with thebranched beta cyclodextrin, the steroid, which is typically waterinsoluble, becomes highly water soluble.

Steroids which are suitable for complexing with branched betacyclodextrins have a molecular structure which allows at least a portionof the steroid's molecular structure to be housed inside the cavity ofthe branched beta cyclodextrin. Suitable steroids includecorticosteroids such as dexamethasone, prednisolone and hydrocortisone;androgens; anabolic steroids; estrogens; and progestagens. Good resultshave been obtained with hydrocortisone, progesterone and testosterone.

In order to form the complex between the branched beta cyclodextrin andthe steroid, an aqueous mixture of steroid and branched betacyclodextrin is prepared and gently mixed at ambient conditions.Stoichiometrically, the ratio of branched beta cyclodextrin to steroidis about 1:1; however, in order to complex as much of the steroid aspossible, preferably an excess of branched beta cyclodextrin is added tothe solution. Most preferably, the stoichiometric ratio of branched betacyclodextrin to steroid is about 2:1, but this will vary with theindividual steroid.

Preferably, first an aqueous solution of branched beta cyclodextrin isprepared such that the solution has a solids concentration of betweenabout 10 to about 50% branched beta cyclodextrin based on the totalweight of solution. Good results have been obtained when the aqueoussolution of branched beta cyclodextrin has a solids concentration ofabout 14% by weight. This mixing is preferably carried out at ambientconditions. In order to prepare this solution, mixing can be employed.To this aqueous solution of branched beta cyclodextrin a steroid isadded in the appropriate amount. The overall mixture of water, branchedbeta cyclodextrin and steroid is then gently mixed by conventional meanswhile maintaining the mixture at ambient conditions. The mixing proceedsuntil an equilibrium is obtained, preferably for about 4 to about 24hours.

The complex of branched beta cyclodextrin and steroid is then obtainedfrom the aqueous solution in a conventional manner, typically byevaporation. Alternatively, the complex is left in solution and merelyconcentrated or evaporated to the appropriate concentration for use.Crystals of G1c7 and hydrocortisone may be obtained by concentration toabout 30% solids. Upon cooling and standing at 30% solids the complexcrystallizes from solution. The material obtained from drying is anequilibrium mixture of complex, free branched beta cyclodextrin and atrace of free steroid. If crystals are obtained, as is the case withG1c7 and hydrocortisone, subsequent solutions of the crystalline complexin water may result in precipitation of free steroid unless anappropriate amount of free branched beta cyclodextrin is present.

Incorporation of the steroid-branched beta cyclodextrin complex in apharmaceutical preparation is accomplished in a conventional manner.However, because of the increased solubility of the complex, the steroidcan more readily be incorporated into a pharmaceutical preparation.

These and other aspects of the present invention may be more fullyunderstood by reference to the following examples.

EXAMPLE 1

This example illustrates preparation of a complex of branched betacyclodextrin and steroid where the steroid is hydrocortisone.

A solution of branched beta cyclodextrin was prepared to about 14%solids by adding branched beta cyclodextrin to water in the amountsshown below in Table 1. To this solution was added the hydrocortisone inthe amounts shown in Table 1 below. The mixture was stirred for about 4hours and then filtered to remove undissolved hydrocortisone. Thefiltrate contained the complex, the free branched beta cyclodextrin andtrace free hydrocortisone. This example was run at ambient conditions.Table 1 below further illustrates this example.

                  TABLE 1                                                         ______________________________________                                        Element                  Amount (gm)                                          ______________________________________                                        Carbohydrate added       1.390                                                Free branched beta cyclodextrin added                                                                  1.307                                                Free branched beta cyclodextrin after                                                                  .413                                                 complexation                                                                  Complexed branched beta cyclodextrin                                                                   .916                                                 Average molecular weight of                                                                            1328                                                 branched beta cyclodextrin                                                    Average DP of branch     1.19                                                 Hydrocortisone complexed in branched                                                                   .250                                                 beta cyclodextrin                                                             Free beta cyclodextrin added (impurity)                                                                .083                                                 Free beta cyclodextrin after complexation                                                              .025                                                 Complexed beta cyclodextrin                                                                            .058                                                 Hydrocortisone complexed in                                                                            .019                                                 beta cyclodextrin                                                             Free hydrocortisone added                                                                              .400                                                 Free soluble hydrocortisone after                                                                      .002                                                 complexation                                                                  Insoluble hydrocortisone after                                                                         .129                                                 complexation                                                                  Complexation hydrocortisone (.271 - 002) =                                                             .269                                                  ##STR1##                                                                     S/S.sub.0 =              79.57                                                Water =                  8.54 ml                                              Solution =               9.46 ml                                              Total Weight of Complex  1.243                                                Total Weight Hydrocortisone: Branched                                                                  1.166                                                beta cyclodextrin                                                             Total Weight Hydrocortisone: Beta                                                                      .077                                                 cyclodextrin                                                                  Efficiency (assume 1:1 complex) .916/1.307 =                                                           .7008                                                ______________________________________                                    

The hydrocortisone was a commercially available hydrocortisone having ahigh purity.

The amount of carbohydrate added, free steroid added, and water weremeasured using a conventional analytical balance. Chromatographicprocedures were used to determine the percentages of branched betacyclodextrin, beta cyclodextrin and other carbohydrates present in thecarbohydrates added. The amount of insoluble steroid after complexationwas determined by the difference between the dry weight of the filterpaper before filtration and the dry weight of the filter paper afterwashing to remove excess carbohydrate and drying. The remaining valuesin the table above were calculated.

The branched beta cyclodextrin was obtained by solvent precipitation andchromatographic separation from a typical CGT digest of 5 DE waxyhydrolysate. The beta cyclodextrin and other carbohydrates which are notbranched beta cyclodextrin are impurities which were not fullyseparated.

The solubility of hydrocortisone in water under ambient conditions hasbeen reported as 0.36 mg/ml. The observed solubility in water of thebranched beta cyclodextrin and hydrocortisone complex in this exampleafter concentration was 65.0 mg/ml under ambient conditions. Byemploying the present invention the solubility of the steroid has beenincreased by a factor of 181.

EXAMPLE 2

This example illustrates preparation of a complex of branched betacyclodextrin and steroid where the steroid is hydrocortisone.

A solution of branched beta cyclodextrin was prepared to about 14%solids by adding branched beta cyclodextrin to water in the amountsshown below in Table 2. To this solution was added the hydrocortisone inthe amounts shown in Table 2 below. The mixture was stirred for about 20hours and then filtered to remove undissolved hydrocortisone. Thefiltrate contained the complex, the free branched beta cyclodextrin andtrace free hydrocortisone. This example was run at ambient conditions.Table 2 below further illustrates this example.

                  TABLE 2                                                         ______________________________________                                        Element                  Amount (gm)                                          ______________________________________                                        Carbohydrate added       1.400                                                Free branched beta cyclodextrin added                                                                  1.202                                                Free branched beta cyclodextrin after                                                                  .361                                                 complexation                                                                  Complexed branched beta cyclodextrin                                                                   .841                                                 Average molecular weight of                                                                            1828                                                 branched beta cyclodextrin                                                    Average DP of branch     4.27                                                 Hydrocortisone complexed in branched                                                                   .167                                                 beta cyclodextrin                                                             Free beta cyclodextrin added (impurity)                                                                --                                                   Free beta cyclodextrin after complexation                                                              --                                                   Complexed beta cyclodextrin                                                                            --                                                   Hydrocortisone complexed in                                                                            --                                                   beta cyclodextrin                                                             Free hydrocortisone added                                                                              .2986                                                Free soluble hydrocortisone after                                                                      .0031                                                complexation                                                                  Insoluble hydrocortisone after complexation                                                            .1286                                                Complexation hydrocortisone (.1700 - .0031) =                                                          .1669                                                 ##STR2##                                                                     S/S.sub.0 =              49.60                                                Water =                  8.6 ml                                               Solution =               9.52 ml                                              Total Weight of Complex  1.008                                                Total Weight Hydrocortisone: Branched                                                                  1.008                                                beta cyclodextrin                                                             Total Weight Hydrocortisone: Beta                                                                      --                                                   cyclodextrin                                                                  ______________________________________                                    

The hydrocortisone was a commercially available hydrocortisone having ahigh purity.

The amount of carbohydrate added, free steroid added, and water weremeasured using a conventional analytical balance. Chromatographicprocedures were used to determine the percentages of branched betacyclodextrin, beta cyclodextrin and other carbohydrates present in thecarbohydrates added. The amount of insoluble steroid after complexationwas determined by the difference between the dry weight of the filterpaper before filtration and the dry weight of the filter paper afterwashing to remove excess carbohydrate and drying. The remaining valuesin the table above were calculated.

The branched beta cyclodextrin was obtained by solvent precipitation andchromatographic separation from a typical CGT digest of 5 DE waxyhydrolysate. The beta cyclodextrin and other carbohydrates which are notbranched beta cyclodextrin are impurities which were not fullyseparated.

The solubility of hydrocortisone in water under ambient conditions hasbeen reported as 0.36 mg/ml. The observed solubility in water of thebranched beta cyclodextrin and hydrocortisone complex in this exampleafter concentration was 21.4 mg/ml under ambient conditions. Byemploying the present invention the solubility of the steroid has beenincreased by a factor of 59.

EXAMPLE 3

This example illustrates preparation of a complex of branched betacyclodextrin and steroid where the steroid is progesterone.

A solution of branched beta cyclodextrin was prepared to about 14%solids by adding branched beta cyclodextrin to water in the amountsshown below in Table 3. To this solution was added the progesterone inthe amounts shown in Table 3 below. The mixture was stirred for about 20hours and then filtered to remove undissolved progesterone. The filtratecontained the complex, the free branched beta cyclodextrin and tracefree progesterone. This example was run at ambient conditions. Table 3below further illustrates this example.

                  TABLE 3                                                         ______________________________________                                        Element                  Amount (gm)                                          ______________________________________                                        Carbohydrate added       1.400                                                Free branched beta cyclodextrin added                                                                  1.202                                                Free branched beta cyclodextrin after                                                                  .677                                                 complexation                                                                  Complexed branched beta cyclodextrin                                                                   .525                                                 Average molecular weight of                                                                            1828                                                 branched beta cyclodextrin                                                    Average DP of branch     4.27                                                 Progesterone complexed in branched                                                                     .0904                                                beta cyclodextrin                                                             Free beta cyclodextrin added (impurity)                                                                --                                                   Free beta cyclodextrin after complexation                                                              --                                                   Complexed beta cyclodextrin                                                                            --                                                   Progesterone complexed in                                                                              --                                                   beta cyclodextrin                                                             Free progesterone added  .3000                                                Free soluble progesterone after complexation                                                           .00014                                               Insoluble progesterone after complexation                                                              .2095                                                Complexation progesterone (.0905 - .0001) =                                                            .0904                                                 ##STR3##                                                                     S/S.sub.0 =              593.5                                                Water =                  8.6 ml                                               Solution =               9.52 ml                                              Total Weight of Complex  .615                                                 Total Weight Progesterone: Branched                                                                    .615                                                 beta cyclodextrin                                                             Total Weight Progesterone: Beta                                                                        --                                                   cyclodextrin                                                                  Efficiency (complexed/total branched                                                                   .437                                                 beta cyclodextrin)                                                            ______________________________________                                    

The progesterone was a commercially available progesterone having a highpurity.

The amount of carbohydrate added, free steroid added, and water weremeasured using a conventional analytical balance. Chromatographicprocedures were used to determine the percentages of branched betacyclodextrin, beta cyclodextrin and other carbohydrates present in thecarbohydrates added. The amount of insoluble steroid after complexationwas determined by the difference between the dry weight of the filterpaper before filtration and the dry weight of the filter paper afterwashing to remove excess carbohydrate and drying. The remaining valuesin the table above were calculated.

The branched beta cyclodextrin was obtained by solvent precipitation andchromatographic separation from a typical CGT digest of 5 DE waxyhydrolysate. The beta cyclodextrin and other carbohydrates which are notbranched beta cyclodextrin are impurities which were not fullyseparated.

The solubility of progesterone in water under ambient conditions hasbeen reported as 0.016 mg/ml. The observed solubility in water of thebranched beta cyclodextrin and progesterone complex in this exampleafter concentration was 44.8 mg/ml under ambient conditions. Byemploying the present invention the solubility of the steroid has beenincreased by a factor of 2800.

EXAMPLE 4

This example illustrates preparation of a complex of branched betacyclodextrin and steroid where the steroid is testosterone.

A solution of branched beta cyclodextrin was prepared to about 14%solids by adding branched beta cyclodextrin to water in the amountsshown below in Table 4. To this solution was added the testosterone inthe amounts shown in Table 4 below. The mixture was stirred for about 20hours and then filtered to remove undissolved testosterone. The filtratecontained the complex, the free branched beta cyclodextrin and tracefree testosterone. This example was run at ambient conditions. Table 4below further illustrates this example.

                  TABLE 4                                                         ______________________________________                                        Element                  Amount (gm)                                          ______________________________________                                        Carbohydrate added       1.400                                                Free branched beta cyclodextrin added                                                                  1.202                                                Free branched beta cyclodextrin after                                                                  .644                                                 complexation                                                                  Complexed branched beta cyclodextrin                                                                   .558                                                 Average molecular weight of                                                                            1828                                                 branched beta cyclodextrin                                                    Average DP of branch     4.27                                                 Testosterone complexed in branched                                                                     .088                                                 beta cyclodextrin                                                             Free beta cyclodextrin added (impurity)                                                                --                                                   Free beta cyclodextrin after complexation                                                              --                                                   Complexed beta cyclodextrin                                                                            --                                                   Testosterone complexed in                                                                              --                                                   beta cyclodextrin                                                             Free testosterone added  .303                                                 Free soluble testosterone after complexation                                                           .00034                                               Insoluble testosterone after complexation                                                              .215                                                 Complexation testosterone (.088 - .000) =                                                              .088                                                  ##STR4##                                                                     S/S.sub.0 =              231.1                                                Water =                  8.6 ml                                               Solution =               9.52 ml                                              Total Weight of Complex  .646                                                 Total Weight Testosterone: Branched                                                                    .646                                                 beta cyclodextrin                                                             Total Weight Testosterone: Beta                                                                        --                                                   cyclodextrin                                                                  Efficiency Complexed: Total Branched                                                                   .464                                                 beta cyclodextrin                                                             ______________________________________                                    

The testosterone was a commercially available testosterone having a highpurity.

The amount of carbohydrate added, free steroid added, and water weremeasured using a conventional analytical balance. Chromatographicprocedures were used to determine the percentages of branched betacyclodextrin, beta cyclodextrin and other carbohydrates present in thecarbohydrates added. The amount of insoluble steroid after complexationwas determined by the difference between the dry weight of the filterpaper before filtration and the dry weight of the filter paper afterwashing to remove excess carbohydrate and drying. The remaining valuesin the table above were calculated.

The branched beta cyclodextrin was obtained by solvent precipitation andchromatographic separation from a typical CGT digest of 5 DE waxyhydrolysate. The beta cyclodextrin and other carbohydrates which are notbranched beta cyclodextrin are impurities which were not fullyseparated.

The solubility of testosterone in water under ambient conditions hasbeen reported as 0.040 mg/ml. The observed solubility in water of thebranched beta cyclodextrin and testosterone complex in this exampleafter concentration was 39.8 mg/ml under ambient conditions. Byemploying the present invention the solubility of the steroid has beenincreased by a factor of 995.

EXAMPLE 5

This example illustrates preparation of a complex of branched betacyclodextrin and steroid where the steroid is hydrocortisone.

A solution of branched beta cyclodextrin was prepared to about 14%solids by adding branched beta cyclodextrin to water in the amountsshown below in Table 5. To this solution was added the hydrocortisone inthe amounts shown in Table 5 below. The mixture was stirred for about 21hours and then filtered to remove undissolved hydrocortisone. Thefiltrate contained the complex, the free branched beta cyclodextrin andtrace free hydrocortisone. This example was run at ambient conditions.Table 5 below further illustrates this example.

                  TABLE 5                                                         ______________________________________                                        Element                  Amount (gm)                                          ______________________________________                                        Carbohydrate added       1.970                                                Free branched beta cyclodextrin added                                                                  1.604                                                Free branched beta cyclodextrin after                                                                  .481                                                 complexation                                                                  Complexed branched beta cyclodextrin                                                                   1.123                                                Average molecular weight of                                                                            1867                                                 branched beta cyclodextrin                                                    Average DP of branch*    4.52                                                 Hydrocortisone complexed in branched                                                                   .218                                                 beta cyclodextrin                                                             Free beta cyclodextrin added (impurity)                                                                .090                                                 Free beta cyclodextrin after complexation                                                              .023                                                 Complexed beta cyclodextrin                                                                            .071                                                 Hydrocortisone complexed in                                                                            .023                                                 beta cyclodextrin                                                             Free hydrocortisone added                                                                              .545                                                 Free soluble hydrocortisone after                                                                      .0044                                                complexation                                                                  Insoluble hydrocortisone after complexation                                                            .299                                                 Complexation hydrocortisone (.246 - .004) =                                                            .242                                                  ##STR5##                                                                     S/S.sub.0 =              51.00                                                Water =                  12.101 ml                                            Solution =               13.40 ml                                             Total Weight of Complex  1.435                                                Total Weight Hydrocortisone: Branched                                                                  1.341                                                beta cyclodextrin                                                             Total Weight Hydrocortisone: Beta                                                                      .094                                                 cyclodextrin                                                                  ______________________________________                                    

The branched beta cyclodextrin was isolated from pyrolyzed branched betacyclodextrin by solvent precipitation and chromatographic separation.The hydrocortisone was a commercially available hydrocortisone having ahigh purity. The amount of carbohydrate added, free steroid added, andwater were measured using a conventional analytical balance.Chromatographic procedures were used to determine the percentages ofbranched beta cyclodextrin, beta cyclodextrin and other carbohydratespresent in the carbohydrates added. The amount of insoluble steroidafter complexation was determined by the difference between the dryweight of the filter paper before filtration and the dry weight of thefilter paper after washing to remove excess carbohydrate and drying. Theremaining values in the table above were calculated.

The solubility of hydrocortisone in water under ambient conditions hasbeen reported as 0.36 mg/ml. The observed solubility in water of thebranched beta cyclodextrin and hydrocortisone complex in this exampleafter concentration was 87.1 mg/ml under ambient conditions. Byemploying the present invention the solubility of the steroid has beenincreased by a factor of 242.

EXAMPLE 6

This example illustrates preparation of a complex of branched betacyclodextrin and steroid where the steroid is hydrocortisone.

A solution of branched beta cyclodextrin was prepared to about 14%solids by adding branched beta cyclodextrin to water in the amountsshown below in Table 6. To this solution was added the hydrocortisone inthe amounts shown in Table 6 below. The mixture was stirred for about 4hours and then filtered to remove undissolved hydrocortisone. Thefiltrate contained the complex, the free branched beta cyclodextrin andtrace free hydrocortisone. This example was run at ambient conditions.Table 6 below further illustrates this example.

                  TABLE 6                                                         ______________________________________                                        Element                  Amount (gm)                                          ______________________________________                                        Carbohydrate added       1.670                                                Free branched beta cyclodextrin added                                                                  1.635                                                Free branched beta cyclodextrin after                                                                  .489                                                 complexation                                                                  Complexed branched beta cyclodextrin                                                                   1.146                                                Average molecular weight of                                                                            4035                                                 branched beta cyclodextrin                                                    Average DP of branch     17.9                                                 Hydrocortisone complexed in branched                                                                   .103                                                 beta cyclodextrin                                                             Free beta cyclodextrin added (impurity)                                                                --                                                   Free beta cyclodextrin after complexation                                                              --                                                   Complexed beta cyclodextrin                                                                            --                                                   Hydrocortisone complexed in                                                                            --                                                   beta cyclodextrin                                                             Free hydrocortisone added                                                                              .150                                                 Free soluble hydrocortisone after                                                                      .004                                                 complexation                                                                  Insoluble hydrocortisone after complexation                                                            .043                                                 Complexation hydrocortisone (.107 - .004) =                                                            .103                                                  ##STR6##                                                                     S/S.sub.0 =              26.07                                                Water =                  10.3 ml                                              Solution =               11.4 ml                                              Total Weight of Complex  1.249                                                Total Weight Hydrocortisone: Branched                                                                  1.249                                                beta cyclodextrin                                                             Total Weight Hydrocortisone: Beta                                                                      --                                                   cyclodextrin                                                                  Efficiency (assume 1:1 complex)                                                                        .7009                                                1.146/1.635                                                                   ______________________________________                                    

The hydrocortisone was a commercially available hydrocortisone having ahigh purity.

The amount of carbohydrate added, free steroid added, and water weremeasured using a conventional analytical balance. Chromatographicprocedures were used to determine the percentages of branched betacyclodextrin, beta cyclodextrin and other carbohydrates present in thecarbohydrates added. The amount of insoluble steroid after complexationwas determined by the difference between the dry weight of the filterpaper before filtration and the dry weight of the filter paper afterwashing to remove excess carbohydrate and drying. The remaining valuesin the table above were calculated.

The branched beta cyclodextrin was obtained by solvent precipitation andchromatographic separation from a typical CGT digest of 5 DE waxyhydrolysate. The beta cyclodextrin and other carbohydrates which are notbranched beta cyclodextrin are impurities which were not fullyseparated.

The solubility of hydrocortisone in water under ambient conditions hasbeen reported as 0.36 mg/ml. The observed solubility in water of thebranched beta cyclodextrin and hydrocortisone complex in this exampleafter concentration was 53.6 mg/ml under ambient conditions. Byemploying the present invention the solubility of the steroid has beenincreased by a factor of 149.

EXAMPLE 7

This example illustrates preparation of a complex of branched betacyclodextrin and steroid where the steroid is cholesterol.

A solution of branched beta cyclodextrin was prepared to about 14%solids by adding branched beta cyclodextrin to water in the amountsshown below in Table 7. To this solution was added the cholesterol inthe amounts shown in Table 7 below. The mixture was stirred for about 20hours and then filtered to remove undissolved cholesterol. The filtratecontained the complex, the free branched beta cyclodextrin and tracefree cholesterol. This example was run at ambient conditions. Table 7below further illustrates this example.

                  TABLE 7                                                         ______________________________________                                        Element                  Amount (gm)                                          ______________________________________                                        Carbohydrate added       1.400                                                Free branched beta cyclodextrin added                                                                  1.202                                                Free branched beta cyclodextrin after                                                                  1.037                                                complexation                                                                  Complexed branched beta cyclodextrin                                                                   .165                                                 Average molecular weight of                                                                            1828                                                 branched beta cyclodextrin                                                    Average DP of branch     4.27                                                 Cholesterol complexed in branched                                                                      .035                                                 beta cyclodextrin                                                             Free beta cyclodextrin added (impurity)                                                                --                                                   Free beta cyclodextrin after complexation                                                              --                                                   Complexed beta cyclodextrin                                                                            --                                                   Cholesterol complexed in --                                                   beta cyclodextrin                                                             Free cholesterol added   .300                                                 Free soluble cholesterol after complexation                                                            .00002                                               Insoluble cholesterol after complexation                                                               .265                                                 Complexation cholesterol (.035 - .000) =                                                               .035                                                  ##STR7##                                                                     S/S.sub.0 =              1838                                                 Water                    8.6 ml                                               Solution =               9.52 ml                                              Total Weight of Complex  .200                                                 Total Weight Cholesterol: Branched                                                                     .200                                                 beta cyclodextrin                                                             Total Weight Cholesterol: Beta                                                                         --                                                   cyclodextrin                                                                  Efficiency =             .137                                                 ______________________________________                                    

The cholesterol was a commercially available cholesterol having a highpurity.

The amount of carbohydrate added, free steroid added, and water weremeasured using a conventional analytical balance. Chromatographicprocedures were used to determine the percentages of branched betacyclodextrin, beta cyclodextrin and other carbohydrates present in thecarbohydrates added. The amount of insoluble steroid after complexationwas determined by the difference between the dry weight of the filterpaper before filtration and the dry weight of the filter paper afterwashing to remove excess carbohydrate and drying. The remaining valuesin the table above were calculated.

The branched beta cyclodextrin was obtained by solvent precipitation andchromatographic separation from a typical CGT digest of 5 DE waxyhydrolysate. The beta cyclodextrin and other carbohydrates which are notbranched beta cyclodextrin are impurities which were not fullyseparated.

The solubility of cholesterol in water under ambient conditions has beenreported as 0,002 mg/ml. The observed solubility in water of thebranched beta cyclodextrin and cholesterol complex in this example was3.7 mg/ml under ambient conditions. By employing the present inventionthe solubility of the steroid has been increased by a factor of 1838.

EXAMPLE 8

This example compares the solubility of the branched betacyclodextrin-steroid complex of the present invention against likesteroids complexed with gamma cyclodextrin, alpha cyclodextrin, betacyclodextrin, and dimethyl beta cyclodextrin. The gamma, alpha, anddimethyl beta cyclodextrin complexes were prepared under ambientconditions with cyclodextrins at a concentration of 0.075M. Because ofthe low solubility of beta cyclodextrin, the concentration of thissolution was 0.0112 M in water under ambient conditions. The results ofthis example are reported in Table 8 below.

                  TABLE 8                                                         ______________________________________                                                Methyl-                                                                       testosterone.sup.(a)                                                                    Solubility (mg/ml)                                                  Testosterone.sup.(b)                                                                    Progesterone                                                                             Hydrocortisone                                   ______________________________________                                        Present   10.03.sup.(b)                                                                             10.32      20.66                                        Invention (Example 4) (Example 3)                                                                              (Example 1)                                  Dimethyl Beta                                                                           13.70.sup.(a)                                                                             13.00      20.30                                        Cyclodextrin                                                                  Gamma     1.40.sup.(a)                                                                              0.09       4.30                                         Cyclodextrin                                                                  Alpha     0.43.sup.(a)                                                                              0.27       3.80                                         Cyclodextrin                                                                  Beta      0.19.sup.(a)                                                                              0.09       1.82                                         Cyclodextrin                                                                  ______________________________________                                    

Clearly the present invention produces superior results compared to thegamma and beta cyclodextrin and comparable results to dimethyl betacyclodextrin.

The values of the dimethyl beta, gamma and alpha cyclodextrin steroidcomplex were taken from reported data.

The values of the present invention reported above were calculated fromExamples 1, 3 and 4 above for comparative purposes.

It will be understood that the preferred embodiments of the presentinvention herein chosen for the purpose of illustration are intended tocover all changes and modifications of the preferred embodiments of thepresent invention which do not constitute a departure from the spiritand scope of the present invention.

What is claimed is:
 1. A water soluble compound formed by complexingbranched beta cyclodextrin with a steroid having a molecular structurethat can fit into the cavity of the branched beta cyclodextrin and forma complex with the branched beta cyclodextrin.
 2. The water solublecompound of claim 1 wherein the steroid is selected from the groupconsisting of: corticosteroids, androgens, anabolic steroids, estrogens,and progestogens; that have a molecular structure that can fit into thecavity of the branched beta cyclodextrin and form a complex with thebranched beta cyclodextrin.
 3. The water soluble compound of claim 1wherein the steroid is selected from the group consisting of:dexamethasone, prednisolone and cholesterol.
 4. The water solublecompound of claim 1 wherein the branched beta cyclodextrin has a branchcontaining between about 1 glucose unit to about 7 glucose units.
 5. Thewater soluble compound of claim 3 wherein the branched beta cyclodextrinhas a branch containing between about 1 glucose unit to about 7 glucoseunits.
 6. A method for increasing the solubility of a steroid comprisingthe steps of:(a) forming an aqueous mixture of branched betacyclodextrin and steroid in a stoichiometric ratio of about 1:1; and (b)recovering a complex of branched beta cyclodextrin and steroid.
 7. Themethod of claim 6 wherein the formation of the aqueous mixture ofbranched beta cyclodextrin and steroid is conducted under ambientconditions.
 8. The method of claim 6 wherein the formation step isaccomplished by adding branched beta cyclodextrin to water and thenadding the steroid and mixing the branched beta cyclodextrin and steroidin water for a period of about 4 to about 24 hours under ambientconditions.
 9. The method of claim 6 wherein the recovery step isaccomplished by evaporation.
 10. The method of claim 8 wherein therecovery step is accomplished by evaporation.